This project seeks to understand how newly synthesized secretory and membrane proteins are made, matured, sorted, and metabolized in cells. This class of proteins is essential to all intercellular and intracellular communication, and their precise locations and abundances are tightly regulated to maintain normal cellular and organismal physiology. Indeed, the majority of current drug targets affect secreted and membrane proteins, underscoring their central role in human biology. Our goals are to develop a molecular level understanding of the pathways of secretory and membrane protein biosynthesis and metabolism. Not only are we interested in these normal cellular events, but also in discovering the ways they are perturbed in certain disease states.[unreadable] [unreadable] Using the biosynthesis of mammalian Prion protein (PrP) as a model system, we have discovered that the most important and tightly regulated step in its initial segregatin to the endoplasmic reticulum (ER) is the interaction between its signal sequence and the protein translocon. This step was found to be critically dependent on a four protein complex of previously unknown function (termed the TRAP complex), in the absence of which PrP does not enter the ER. Our finding that not all signal sequences require TRAP suggests that different substrates are recognized differently by the translocon, an idea further supported by recent studies analyzing crosslinking between signal sequences and translocon components.[unreadable] [unreadable] More significantly, we have now shown that alterations in the nature of this signal-translocon interaction have substantial consequences for protein localization and function. In the case of PrP, the cellular burden of potentially cytotoxic forms can be reduced (or enhanced) to change the susceptibility of cells to otherwise harmful insults. In the case of another protein, Calreticulin, we find that signal-translocon interactions are critical in allowing this protein to exist in two compartments (the ER lumen and the cytosol), where it serves independent functions. Thus, advances during the past year are beginning to illuminate a novel site of potential cellular regulation, the entry of secretory and membrane protein substrates into the mammalian secretory pathway, that impacts both normal physiology and disease progression. Most recently, these insights have been applied to uncover the ways in which protein entry into the ER is modulated productively by the cell under conditions of stress. This analysis has led to the discovery of a new degradation pathway we have termed pre-emptive quality control (or pQC). The mechanisms that facilitate pQC and the degradative machinery are currently being studied.[unreadable] [unreadable] In parallel collaborative studies, we are using physiological, structural, and pharmacological approaches to understand components of the protein translocation machinery at the mammalian ER. In the physiological approach, we are examining the role of cytosolic calreticulin generated by translocation regulation in mice models. In the structural approach, we are applying cryo-electron microscopy to visualize intact ribosome-translocon complexes. By preparing and analyzing translocon complexes lacking or containing specific components such as the TRAP complex, we are able to determine the relative positions of the various proteins comprising the translocon. In the pharmacologic approach, we are utilizing novel assays for translocation to identify, characterize, and study small molecule inhibitors of protein translocation. The goal of these studies is to develop probes that facilitate the modulation of protein translocation in vivo to understand the role of this process in normal and pathological cellular physiology.[unreadable] [unreadable] We are also performing a systematic analysis of the biosynthesis, trafficking, and metabolism of disease-associated PrP mutants. The aim of these studies is to identify precisely the cellular locale and mechanism of PrP misfolding that initiates the disease process. Our current analyses have narrowed the event to a post-ER location, a finding that is notable because it is after the principal site of cellular quality control used by secretory and membrane proteins. In parallel studies, the downstream consequences of PrP misfolding and aggregation are being studied to identify the mechanism by which these events lead to cellular dysfunction. We have now found that these aggregates recruit various cellular factors, therby depleting their functional availability. One such factor is of particular importance because its disruption in mice leads directly to a neurodegenerative phenotype reminiscent of diseases caused by PrP. [unreadable] [unreadable] And finally, we have been investigating the molecular mechanisms and machinery for the insertion of membrane proteins into the ER membrane. We have now identified a novel targeting factor that is highly conserved, broadly expressed, and plays a key role in the insertion of a large class of physiologically important membrane proteins called 'tail-anchored' proteins. This factor appears to interact with nascent tail-anchored membrane protein substrates in the cytosol and delivers them to a yet unidentified receptor at the ER membrane. The mechanisms by which this factor operates and the identification of additional components in this pathway are currently under investigation.